基于纳米抗体-碱性磷酸酶融合蛋白的高灵敏度直接竞争荧光酶免疫分析方法的建立及其用于尿液中 3-苯氧苯甲酸的检测。
Development of a Highly Sensitive Direct Competitive Fluorescence Enzyme Immunoassay Based on a Nanobody-Alkaline Phosphatase Fusion Protein for Detection of 3-Phenoxybenzoic Acid in Urine.
机构信息
College of Plant Protection , Agricultural University of Hebei , Baoding 071001 , PR China.
Department of Entomology and Nematology and UCD Comprehensive Cancer Center , University of California , Davis , California 95616 , United States.
出版信息
J Agric Food Chem. 2018 Oct 31;66(43):11284-11290. doi: 10.1021/acs.jafc.8b04521. Epub 2018 Oct 17.
Abstract
3-Phenoxybenzoic acid (3-PBA) is a human urinary metabolite of many pyrethroid insecticides and can be used as a biomarker to monitor human exposure to these pesticides. A rapid and sensitive direct competitive fluorescence enzyme immunoassay (dc-FEIA) for detecting 3-PBA on the basis of a nanobody (Nb)-alkaline phosphatase (AP) fusion protein was developed. The anti-3-PBA Nb-AP fusion protein was expressed and purified. The 50% inhibitory concentration (IC) and linear range of dc-FEIA were 0.082 and 0.015-0.447 ng/mL, respectively, with a detection limit of 0.011 ng/mL. The IC of dc-FEIA was improved by nearly ten times compared with those of one-step and three-step direct competitive enzyme-linked immunosorbent assay (dc-ELISA). Spiked urine samples were detected by both dc-FEIA and liquid chromatography-mass spectrometry (LC-MS), and the results showed good consistency between the two analysis methods, indicating the reliability of dc-FEIA based on the Nb-AP fusion protein for detecting 3-PBA in urine.
摘要
3-苯氧基苯甲酸（3-PBA）是许多拟除虫菊酯类杀虫剂在人体尿液中的代谢物，可用作监测人体接触这些农药的生物标志物。基于纳米抗体（Nb）-碱性磷酸酶（AP）融合蛋白，建立了一种快速灵敏的直接竞争荧光酶免疫分析（dc-FEIA）方法来检测 3-PBA。表达并纯化了抗 3-PBA Nb-AP 融合蛋白。dc-FEIA 的 50%抑制浓度（IC）和线性范围分别为 0.082 和 0.015-0.447ng/mL，检测限为 0.011ng/mL。与一步法和三步法直接竞争酶联免疫吸附测定（dc-ELISA）相比，dc-FEIA 的 IC 提高了近十倍。用 dc-FEIA 和液相色谱-质谱（LC-MS）检测了加标尿液样品，两种分析方法的结果具有良好的一致性，表明基于 Nb-AP 融合蛋白的 dc-FEIA 用于检测尿液中 3-PBA 的可靠性。
相似文献
1
Development of a Highly Sensitive Direct Competitive Fluorescence Enzyme Immunoassay Based on a Nanobody-Alkaline Phosphatase Fusion Protein for Detection of 3-Phenoxybenzoic Acid in Urine.基于纳米抗体-碱性磷酸酶融合蛋白的高灵敏度直接竞争荧光酶免疫分析方法的建立及其用于尿液中 3-苯氧苯甲酸的检测。
J Agric Food Chem. 2018 Oct 31;66(43):11284-11290. doi: 10.1021/acs.jafc.8b04521. Epub 2018 Oct 17.
2
Investigation of the Small Size of Nanobodies for a Sensitive Fluorescence Polarization Immunoassay for Small Molecules: 3-Phenoxybenzoic Acid, an Exposure Biomarker of Pyrethroid Insecticides as a Model.小分子灵敏荧光偏振免疫分析中小纳米体的尺寸研究：以 3-苯氧基苯甲酸（拟除虫菊酯杀虫剂的暴露生物标志物）为模型。
J Agric Food Chem. 2019 Oct 16;67(41):11536-11541. doi: 10.1021/acs.jafc.9b04621. Epub 2019 Oct 7.
3
Development of a nanobody-alkaline phosphatase fusion protein and its application in a highly sensitive direct competitive fluorescence enzyme immunoassay for detection of ochratoxin A in cereal.纳米抗体-碱性磷酸酶融合蛋白的研制及其在谷物中赭曲霉毒素A高灵敏度直接竞争荧光酶免疫分析中的应用。
Anal Chem. 2015 Jan 20;87(2):1387-94. doi: 10.1021/ac504305z. Epub 2015 Jan 6.
4
Anti-idiotypic nanobody-alkaline phosphatase fusion proteins: Development of a one-step competitive enzyme immunoassay for fumonisin B1 detection in cereal.抗独特型纳米抗体-碱性磷酸酶融合蛋白：用于谷物中伏马菌素B1检测的一步竞争性酶免疫测定法的开发。
Anal Chim Acta. 2016 Jun 14;924:53-59. doi: 10.1016/j.aca.2016.03.053. Epub 2016 Apr 7.
5
Multivalent nanobody as capture antibody-based enzyme linked immunosorbent assay for detection of 3-phenoxybenzoic acid in urine.基于多价纳米抗体捕获抗体的酶联免疫吸附测定法检测尿液中的3-苯氧基苯甲酸
Anal Biochem. 2021 Nov 1;632:114390. doi: 10.1016/j.ab.2021.114390. Epub 2021 Sep 21.
6
Isolation of Bactrian Camel Single Domain Antibody for Parathion and Development of One-Step dc-FEIA Method Using VHH-Alkaline Phosphatase Fusion Protein.利用 VHH-碱性磷酸酶融合蛋白分离双峰驼单域抗体对对硫磷，并建立一步 dc-FEIA 方法。
Anal Chem. 2018 Nov 6;90(21):12886-12892. doi: 10.1021/acs.analchem.8b03509. Epub 2018 Oct 12.
7
An Innovative Nanobody-Based Electrochemical Immunosensor Using Decorated Nylon Nanofibers for Point-of-Care Monitoring of Human Exposure to Pyrethroid Insecticides.一种基于创新纳米抗体的电化学免疫传感器，使用修饰的尼龙纳米纤维用于现场监测人体接触拟除虫菊酯类杀虫剂。
ACS Appl Mater Interfaces. 2020 Feb 5;12(5):6159-6168. doi: 10.1021/acsami.9b16193. Epub 2020 Jan 23.
8
Development of nanobody-based flow-through dot ELISA and lateral-flow immunoassay for rapid detection of 3-phenoxybenzoic acid.基于纳米抗体的流通式斑点酶联免疫吸附测定法和侧向流动免疫分析法用于快速检测3-苯氧基苯甲酸的研究进展
Anal Methods. 2021 Apr 14;13(14):1757-1765. doi: 10.1039/d1ay00129a. Epub 2021 Mar 24.
9
Development of a single-chain variable fragment-alkaline phosphatase fusion protein and a sensitive direct competitive chemiluminescent enzyme immunoassay for detection of ractopamine in pork.研制单链可变片段-碱性磷酸酶融合蛋白，并建立一种用于检测猪肉中莱克多巴胺的灵敏直接竞争化学发光酶免疫分析方法。
Anal Chim Acta. 2012 Jul 29;736:85-91. doi: 10.1016/j.aca.2012.05.033. Epub 2012 May 28.
10
Biological monitoring of 3-phenoxybenzoic acid in urine by an enzyme-linked immunosorbent assay.尿液中 3-苯氧基苯甲酸的酶联免疫吸附测定法生物监测。
Talanta. 2011 Feb 15;83(5):1317-23. doi: 10.1016/j.talanta.2010.07.077. Epub 2010 Aug 6.
引用本文的文献
1
Multifunctional Nanobody Fusion Proteins in Immunoassays: Diverse Strategies for Enhanced Analytical Performance.免疫分析中的多功能纳米抗体融合蛋白：提高分析性能的多种策略。
Trends Analyt Chem. 2025 Nov;192. doi: 10.1016/j.trac.2025.118404. Epub 2025 Aug 2.
2
Overview on the Development of Alkaline-Phosphatase-Linked Optical Immunoassays.碱性磷酸酶连接光学免疫分析的发展综述
Molecules. 2023 Sep 11;28(18):6565. doi: 10.3390/molecules28186565.
3
Recent Advances in the Recognition Elements of Sensors to Detect Pyrethroids in Food: A Review.近年来用于检测食品中拟除虫菊酯的传感器识别元件的研究进展：综述。
Biosensors (Basel). 2022 Jun 10;12(6):402. doi: 10.3390/bios12060402.
4
Generation of Dual functional Nanobody-Nanoluciferase Fusion and its potential in Bioluminescence Enzyme Immunoassay for trace Glypican-3 in Serum.双功能纳米抗体-纳米荧光素酶融合蛋白的构建及其在血清中痕量磷脂酰肌醇蛋白聚糖-3生物发光酶免疫分析中的应用潜力
Sens Actuators B Chem. 2021 Jun 1;336. doi: 10.1016/j.snb.2021.129717. Epub 2021 Mar 2.
5
Smartphone-Based Dual-Channel Immunochromatographic Test Strip with Polymer Quantum Dot Labels for Simultaneous Detection of Cypermethrin and 3-Phenoxybenzoic Acid.基于智能手机的带有聚合物量子点标记的双通道免疫层析试纸条，用于同时检测氯氰菊酯和3-苯氧基苯甲酸。
Anal Chem. 2021 Oct 12;93(40):13658-13666. doi: 10.1021/acs.analchem.1c03085. Epub 2021 Sep 30.
6
Application of Single-Domain Antibodies ("Nanobodies") to Laboratory Diagnosis.单域抗体（“纳米抗体”）在实验室诊断中的应用。
Ann Lab Med. 2021 Nov 1;41(6):549-558. doi: 10.3343/alm.2021.41.6.549.
7
Development of nanobody-based flow-through dot ELISA and lateral-flow immunoassay for rapid detection of 3-phenoxybenzoic acid.基于纳米抗体的流通式斑点酶联免疫吸附测定法和侧向流动免疫分析法用于快速检测3-苯氧基苯甲酸的研究进展
Anal Methods. 2021 Apr 14;13(14):1757-1765. doi: 10.1039/d1ay00129a. Epub 2021 Mar 24.
8
An Ultrasensitive Bioluminescent Enzyme Immunoassay Based on Nanobody/Nanoluciferase Heptamer Fusion for the Detection of Tetrabromobisphenol A in Sediment.基于纳米抗体/纳米荧光素酶七聚体融合的超灵敏生物发光酶免疫分析用于沉积物中四溴双酚 A 的检测。
Anal Chem. 2020 Jul 21;92(14):10083-10090. doi: 10.1021/acs.analchem.0c01908. Epub 2020 Jul 7.
9
An Innovative Nanobody-Based Electrochemical Immunosensor Using Decorated Nylon Nanofibers for Point-of-Care Monitoring of Human Exposure to Pyrethroid Insecticides.一种基于创新纳米抗体的电化学免疫传感器，使用修饰的尼龙纳米纤维用于现场监测人体接触拟除虫菊酯类杀虫剂。
ACS Appl Mater Interfaces. 2020 Feb 5;12(5):6159-6168. doi: 10.1021/acsami.9b16193. Epub 2020 Jan 23.
10
A rapid and simple fluorescence enzyme-linked immunosorbent assay for tetrabromobisphenol A in soil samples based on a bifunctional fusion protein.基于双功能融合蛋白的土壤样品中四溴双酚 A 的快速简便荧光酶联免疫吸附测定法。
Ecotoxicol Environ Saf. 2020 Jan 30;188:109904. doi: 10.1016/j.ecoenv.2019.109904. Epub 2019 Nov 6.
本文引用的文献
1
Pyrethroid Pesticide Exposure and Risk of Primary Ovarian Insufficiency in Chinese Women.拟除虫菊酯类农药暴露与中国女性原发性卵巢功能不全风险。
Environ Sci Technol. 2018 Mar 6;52(5):3240-3248. doi: 10.1021/acs.est.7b06689. Epub 2018 Feb 22.
2
Phage-Mediated Competitive Chemiluminescent Immunoassay for Detecting Cry1Ab Toxin by Using an Anti-Idiotypic Camel Nanobody.利用抗独特型骆驼纳米抗体的噬菌体介导竞争性化学发光免疫分析法检测Cry1Ab毒素
J Agric Food Chem. 2018 Jan 31;66(4):950-956. doi: 10.1021/acs.jafc.7b04923. Epub 2018 Jan 22.
3
Pyrethroids inhibit K2P channels and activate sensory neurons: basis of insecticide-induced paraesthesias.拟除虫菊酯抑制 K2P 通道并激活感觉神经元：杀虫剂诱导感觉异常的基础。
Pain. 2018 Jan;159(1):92-105. doi: 10.1097/j.pain.0000000000001068.
4
Simultaneous determination of pesticide residues and antioxidants in blended oil using a liquid-liquid extraction combined with dispersive solid phase extraction method.采用液液萃取结合分散固相萃取法同时测定混合油中的农药残留和抗氧化剂。
Food Chem. 2017 Aug 15;229:347-353. doi: 10.1016/j.foodchem.2017.02.094. Epub 2017 Feb 21.
5
Pyrethroid insecticide lambda-cyhalothrin and its metabolites induce liver injury through the activation of oxidative stress and proinflammatory gene expression in rats following acute and subchronic exposure.拟除虫菊酯类杀虫剂高效氯氟氰菊酯及其代谢产物在大鼠急性和亚慢性暴露后，通过激活氧化应激和促炎基因表达诱导肝损伤。
Environ Sci Pollut Res Int. 2017 Feb;24(6):5841-5856. doi: 10.1007/s11356-016-8323-4. Epub 2017 Jan 6.
6
Home Use of a Pyrethroid-Containing Pesticide and Facial Paresthesia in a Toddler: A Case Report.幼儿家用拟除虫菊酯类杀虫剂与面部感觉异常：一例报告
Int J Environ Res Public Health. 2016 Aug 17;13(8):829. doi: 10.3390/ijerph13080829.
7
Detection of 3-phenoxybenzoic acid in river water with a colloidal gold-based lateral flow immunoassay.基于胶体金的侧向流动免疫分析法检测河水中的3-苯氧基苯甲酸
Anal Biochem. 2015 Aug 15;483:7-11. doi: 10.1016/j.ab.2015.04.022. Epub 2015 May 5.
8
One-step immunoassay for tetrabromobisphenol a using a camelid single domain antibody-alkaline phosphatase fusion protein.使用骆驼科单域抗体-碱性磷酸酶融合蛋白的四溴双酚A一步免疫测定法。
Anal Chem. 2015;87(9):4741-8. doi: 10.1021/ac504735p. Epub 2015 Apr 22.
9
Pyrethroids: exposure and health effects--an update.拟除虫菊酯：暴露与健康影响——最新情况
Int J Hyg Environ Health. 2015 May;218(3):281-92. doi: 10.1016/j.ijheh.2015.01.002. Epub 2015 Jan 15.
10
Developmental pesticide exposure reproduces features of attention deficit hyperactivity disorder.发育期接触杀虫剂会重现注意力缺陷多动障碍的特征。
FASEB J. 2015 May;29(5):1960-72. doi: 10.1096/fj.14-260901. Epub 2015 Jan 28.
Zotero 插件